Effect of Australian & Malaysian originated Daucus carota on the concentration of β-carotene.

To what extent does the Australian and Malaysian originated carrots (Daucus carota) affect the concentrations of β-carotene present by extracting β-carotene from the Daucus carota from using solvent extraction and measuring the absorbance of β-carotene using a UV Spectrophotometer, followed by calculating the concentration of β-carotene present in both origins of Daucus carota?

The consumption of vegetables over the years has been increasing tremendously due to the myriad of people changing their lifestyles in order to lead healthier lives. Furthermore, the consumption of organically-produced foods has been drastically increasing due to the attention it obtains from social media. Thus, there is a paradigm shift in terms of food choices as now, more people are consuming healthier foods. According to a survey done by The Research Institute of Organic Agriculture and the International Federation of Organic Agriculture Movements in 2015, it showed that the growth in the market for organic foods (vegetables) had increased almost five-fold since 1999 [15.2 bn $USD] to 2013 [72.0 bn $USD] (Willer). This statistic supports the fact that the consumption of vegetables is ever growing.

Australian farms predominantly grow their vegetables organically. This means that about 25 pesticides are only allowed to be used to produce organic foods as compared to conventional production in Malaysia where over 900 pesticides can be used (Roseboro). Additionally, This is also another contributing factor to why so many people prefer organically-produced food over conventionally grown foods as it is a healthier option. However, the price allocated to organically-produced foods is considerably higher than conventionally grown foods. I wanted to see whether the concentration  β-carotene correlated with the origin of the Daucus carota (CABI) itself. Moreover, I wanted to know why my parents would rather buy carrots from a specific origin such as Australia, even when given the high price?

β-carotene is the orange pigment which is found in photosynthetic organisms. It is instrumental  in photosynthesis, and is an accessory pigment in the light-dependent reaction. It is also an antenna pigment due to its properties, enabling it to form protein complexes such as photosystems that absorb photons of light (Andrew Allot).  β-carotene’s main function is to essentially defend the plant from molecular oxidation from singlet oxygen which are produced from chlorophyll triplet states. Singlet oxygen is a highly unstable molecule produced during photosynthesis and can lead to oxidation or isomerisation of the plant. Oxidation or isomerisation could hinder the process of photosynthesis in the plant.

UV-Spectrophotometry will be used to determine the concentration of  β-carotene present in the Daucus carota. It essentially measures the absorbance of a particular solution in order to determine the concentration of  β-carotene. Based on the absorption spectrum of  β-carotene,  β-carotene absorbs the most light around 410nm-490nm(Evens). Hence, the colour region would be calibrated to the green-blue colour region according to the respective wavelength chosen, in this case being 450nm. Once the absorbance of  β-carotene is determined, the concentration of  β-carotene can be calculated using

\(\frac{A×V(ml)\times10^4}{A^{1\%}_ {1cm}\times \ P\ (g)}\)

A refers the absorbance used [450nm], V refers to the volume of β-carotene extracted. P refers to the weight of Daucus carota used and \(A^{1\%}_ {1cm}\)  represents the β-carotene coefficient [2500] (Cucurbita Moschata Duch).

Organic farming uses a higher concentration of potassium/K⁺ ions as compared to conventional farming methods (Fess ID, Benedito). The increase in K⁺ ions correlates with the increase in ATP [Adenine tri-phosphate] formation during aerobic respiration in the plant. More ATP corresponds to a higher rate of hypertrophy, which increases the size of the organelles. Therefore, having an enlarged cell would allow for a higher concentration of β-carotene to occupy the thylakoid membranes of the chloroplasts (Bogacz-Radomska, Harasym). Furthermore, a study indicated that photosynthetic rate can also be affected by the concentration of metal ions present in the air (Tarek Houri, Yara Khirallah, et al). This would lead to the decline in the concentration of β-carotene as metal ions and primary pollutants attack the chloroplast, leading to the destruction of its organelles (Sewelam, et al). Since β-carotene is located in the thylakoid membrane, it would also be adversely affected. Metal pollution is determined to be higher in Malaysia than in Australia (Bernhard A, et al), (Laidlaw, et al). Therefore, this leads to the hypothesis.

H₁:The Australian Daucus carota will contain a higher concentration of β-carotene as compared to Malaysian Daucus carota.

H₀: There is no statistical significance between the concentrations of β-carotene between the Malaysian and Australian originated Daucus Carota.

In order to obtain the concentration of the β-carotene, the process will be needed to be split into 3 separate sections. The first process requires the extraction of carotene from the carrot. This is followed by measuring the absorbance of the carotene. Finally, the absorbance of all the values will be used to calculate the concentration of β-carotene. A t-test is then used in order to evaluate the accuracy of the data (Rodriguez-Amaya).

• Measure 10.00 grams of Australian Daucus carota using an electronic balance.
• Place 10.00 grams of Australian Daucus carota into a mortar.
• Start to crush the Daucus carota into tiny pieces using the pestle.
• Once the Australian Daucus carota has been crushed, pour 25.0 cm³ of acetone using a 25.0 cm³ measuring cylinder into the mortar and further crush the Australian Daucus carota.
• After a few minutes, pour 25.0 cm³ of hexane using a 25.0 cm³ measuring cylinder into each mortar and start to further crush the carrots.
• After 10.0 minutes, pour an additional 30 cm³ of acetone using a 50 cm³ measuring cylinder into the mortar and start to crush again. At this point, the Australian Daucus carota should be close to being completely macerated.
• Stop crushing the Australian Daucus carota once an orange solution is finally formed in the mortar.
• Use a 50 cm³ gas syringe, transfer the solution from the mortar into separate 500 cm³ separating funnels. The solutions in the separating funnels have to be filtered through a filter paper first. This is to get rid of the macerated Australian Daucus carota.
• Next, prepare 100 cm³ of 10% sodium chloride solution by diluting 10.00 grams of pure solidified sodium chloride followed by adding distilled water until it reaches the 100 cm³ mark on the 100 cm³ measuring cylinder.
• Pour 100 cm³ of distilled water into each separating funnel and shake the mixture.
• This is then followed by adding 100 cm³ of 10% sodium chloride solution to the separating funnel. The NaCl salt is added to the mixture to allow for the cells to rupture and release β-carotene.
• Shake the separating funnel vigorously and wait for approximately 5.0 minutes for the liquids to settle and separate. Remember to open the stopcock while shaking to release pressure in the flask. After shaking, 2 layers should be observed. The top layer is the supernatant containing β-carotene.
• Open the stopcock to allow the bottom layer of the mixture containing water and sodium chloride to flow out into a measuring cylinder and close the stopcock once the supernatant is only left in the separating funnel.
• The supernatant remaining in the separating funnel contains the β-carotene (Rebecca)
• Repeat steps 1 to 14 for the Malaysian originated Daucus carota

• Once the β-carotene has been extracted, use a UV Spectrophotometer at a wavelength of 450 nm (Lucia). to determine the absorbance of the β-carotene. Do this for both solutions from their respective separating funnels.
• Once the absorbance of the carotenoids is determined, use the formula to calculate the 

concentration of β-carotene,\(\frac{A×V(ml)\times10^4}{A^{1\%}_{1cm} × P \ (g)}\).

Where A: Absorbance reading found at 450nm

V: Total extracted volume of β-carotene in mL

\(A^{1\%}_{1cm}\) β-carotene coefficient at 2500

P: Sample weight in grams

A T-test will also be used. This is to determine the statistical significance as well as determine whether there is a significant difference in terms of their β-carotene concentrations.


The formula is: t = \(\frac{(x_1-x_2)}{\sqrt{\frac{(s_1)^2}{n_1}+\frac{(s_2)^2}{n_2}}}.\) x₁ and x₂ is the mean of the sample sizes of each origin of Daucus carota(Australian and Malaysian). s₁ and s₂ refer to the standard deviation of each sample while n is the sample size of each origin.

• Since the use of a knife is prevalent and it has a sharp edge, take caution whilst cutting the carrots.
• Hexane is an organic solvent, thus short-term exposure could cause nausea, drowsiness and even unconsciousness. Long-term exposure could potentially cause neurotoxic damage (MSDS).
• The use of a lab coat was ubiquitous as hexane and acetone could easily spill causing irritation or even corrosion of the skin.
• Use the fume hood when dealing with chemicals like hexane as they are carcinogenic  (MSDS).

• The carrots that were not used for the experiment can be composted.
• A minimal volume of chemicals were used to prevent any further wastage. However, this was done through trial and error to ensure the most volume of carotenoids with the least volume of solvents used.

• Hexane once again has to be disposed properly as if it is mishandled and thrown into the sink, it could potentially end up in bodies of water and this could indirectly affect marine life. One way to tackle this problem is by disposing the hexane into an organic waste bottle.

• As hexane and acetone were added, it became easier to crush the Daucus carota with a pestle.
• After the maceration, the solution possessed a dark orange colour.
• As the solution was filtered through a filter paper, the Daucus carota pieces were observed as the residue.
• When the sodium chloride solution together with water was added into the separating funnel, the mixture was shaken vigorously for 5.0 minutes. Every time the stopcock was opened, a loud hissing sound was made.
• After shaking the separating funnel and letting the mixture settle, 2 distinguishable layers had formed that were separated by a white mucus layer that separated the 2 layers. 
• The extracted β-carotene possessed a dark orange colour.